EP0231816A2 - Pharmaceutical preparations for the stabilization of interferon alpha - Google Patents

Pharmaceutical preparations for the stabilization of interferon alpha Download PDF

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Publication number
EP0231816A2
EP0231816A2 EP87100792A EP87100792A EP0231816A2 EP 0231816 A2 EP0231816 A2 EP 0231816A2 EP 87100792 A EP87100792 A EP 87100792A EP 87100792 A EP87100792 A EP 87100792A EP 0231816 A2 EP0231816 A2 EP 0231816A2
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Prior art keywords
pharmaceutical preparation
gelatin
interferon alpha
preparation forms
forms according
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EP87100792A
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German (de)
French (fr)
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EP0231816B1 (en
EP0231816A3 (en
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Helmut Dr. Franz
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Boehringer Ingelheim Pharma GmbH and Co KG
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Dr Karl Thomae GmbH
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions

Definitions

  • interferon alpha interferon beta and interferon gamma, corresponding to IFN alpha, IFN beta, IFN gamma.
  • the synthesis site of IFN alpha to which the present invention relates are the leucocytes which, after stimulation or induction by appropriate inducers (e.g. viruses or poly I ⁇ poly C)), synthesize and release IFN alpha.
  • Bacteria transformed with suitable DNA sequences can synthesize IFN alpha, which have a biological activity analogous to the real human IFN alpha, with the advantage that highly purified and essentially contaminant-free proteins can be obtained in comparatively large amounts, which are suitable for clinical use , for example against various virus infections and malignancies.
  • the application route is of particular importance with regard to the quality of effects and the rate of side effects.
  • the disadvantages are particularly impressive when it comes to the systemic use of interferons, since they reach and influence not only the target tissue, but also healthy, uninfected or untransformed cells or organs and therefore produce side effects that can prevent intravenous or intramuscular use.
  • the systemic application did not always show the desired effectiveness.
  • a direct injection may be possible, the agent of choice, e.g. B. for virus - or tumor - related malignancies or other diseases which can be treated with interferon and which are accessible to external and local treatment (e.g. skin, eyes, nose), but is rather the topical application of interferon or IFN alpha, on which this invention relates.
  • IFN alpha and interferons in general is associated with considerable difficulties from various points of view. From the point of view of the pharmaceutical formulation, it must be demanded that the biologically active IFN alpha obtains a sufficiently high stability to act both at room temperature and at body temperature for a sufficiently long time or over a suitable period of several hours "in situ" to remain effective. It is known that IFN alpha a very temperature-unstable protein. Significant activity losses of up to 80% were observed within a few weeks at 4 ° C.
  • the necessary stability-maintaining components must be such that they neither cause intolerance reactions at the application site, conceivable through the action of biologically active foreign substances, nor impair the penetration of IFN alpha or prevent diffusion.
  • hydroxyethyl cellulose is a most suitable agent for the production of gels of this or other type, e.g. B. because of its heat stability, is present and is commercially available.
  • hydroxyethyl cellulose for the production of an IFN alpha-containing matrix compared to other carriers is that it does not coagulate or gel at the boiling point of water, is readily water-soluble, and that the viscosity of such a solution increases with decreasing temperature.
  • Terano has described stabilization of interferon in aqueous solution only on the basis of a chemically modified gelatin, also for a pharmaceutical interferon preparation to be injected, in order to substitute human serum albumin as a known interferon-stabilizing agent for injection (EP- A-0 l62 332). It has long been known to the person skilled in the art that gelatin per se has a stabilizing effect on interferons (Sedmak JJ and Grossberg, SE Tex. Rep. Biol. Med. 4l, 274-279, l982), and that gelatin and chemically modified derivatives thereof exert a certain stabilizing effect on some other biological proteins (eg DE-Al ll8 732, EP-A-0 l56 242).
  • sugar alcohols such as glycerol, erythoritol, arabitol, xylitol, sorbitol and mannitol
  • sugar acids such as iduronic acid, galacturonic acid and other uronic acids, ascorbic acid and their salts
  • organic buffer systems such as citrate buffer, succinate Buffer, tartrate buffer, fumarate buffer, glyconate buffer, oxalate buffer, lactate buffer and acetate buffer have been disclosed in connection with various pharmaceutical carriers such as waxes, sodium carboxymethyl cellulose, carboxyvinyl derivatives and water (EP-A-0 080 879).
  • the object of the present invention was to find a carrier matrix for highly purified interferon alpha, especially for recombinantly produced IFN alpha, which at least meet the requirements enumerated above and, on the other hand, is as cheap as possible both from a manufacturing and economic point of view, i.e. simple composition, should have, without a loss of a required optimal effectiveness of the biologically active active ingredient IFN alpha contained in the carrier matrix for topical application.
  • the present invention achieved this complex of problems by combining hydroxyethyl cellulose and gelatin in certain mixing ratios and thus creating a carrier and stabilizer for IFN alpha which not only meets the requirements described above, but surprisingly also makes IFN alpha particularly stable gives biological activity and is therefore ideally suited for the topical application of highly purified IFN alpha, in particular produced using recombinant DNA technology.
  • the carrier / stabilizer complex found is free of human serum Albumin is, which is known to lead to local immune reactions and allergies, especially when used topically, especially over longer periods of time.
  • the present invention therefore relates to an improved pharmaceutical formulation for the topical application of IFN alpha.
  • the two individual components acting as carrier and stabilizer namely hydroxyethyl cellulose and gelatin, exert an activity-preserving effect on IFN alpha which goes beyond what is to be expected, taking into account the fact that IFN alpha and interferons are generally known for eg against physical influences to be unstable proteins - both in "naked” form and incorporated in other gel matrices (e.g.
  • the pharmaceutical preparation forms for IFN alpha according to the invention which are to be applied topically, are preferably used as virus static agents, the therapeutic use in the dermal, nasal, ophthalmic and intravaginal area, for example for herpes labialis, genital herpes including papilloma infections, warts and herpes zoster infections , he follows.
  • the scope of application also includes the topical treatment of paracanceroses and canceroses.
  • This invention relates to topical pharmaceutical preparation forms with a stabilization of the active ingredient contained therein, human interferon alpha (IFN alpha), consisting of l. a carrier and stabilizer in the complex for the active ingredient, 2. an antiadhesive and 3. if appropriate, other customary active ingredients such as buffers and salts, humectants, preservatives, penetration promoters, vehicles.
  • IFN alpha human interferon alpha
  • cellulose derivatives carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose
  • polyvinyl alcohols polyvinyl pyrrolidone
  • polyacrylic acid polymers Carbopol® and mixtures thereof together with gelatin, acid or alkaline digested
  • Preferred carriers and stabilizers for IFN alpha uses hydroxyethyl cellulose with a degree of substitution of 2.5 in certain mixing ratios of 0.1 to 2% by weight and gelatin of 0.1 to 10% by weight, based in each case on the finished product, using polyvinyl alcohols and polyvinyl pyrrolidone , up to 15% by weight of this is used, thus achieving a special stabilization especially of IFN alpha, as is also demonstrated by the tests carried out on the stability behavior and how the curves shown in Fig. 1 to 3, which have a test period of 14 weeks for storage repairs each include from -l0, +4 and + 2l ° C, show an example.
  • Anionic, cationic and neutral surface-active substances can be used as anti-adhesive agents for better spreading of the gels and as anti-adhesive filtration aid, preferably polyethoxylated sorbitan esters such as polysorbate 20, 60, 80 are used.
  • polyethoxylated sorbitan esters such as polysorbate 20, 60, 80 are used.
  • inorganic and organic buffer solutions with an IFN alpha - Preparations optimal pH in the range of 6.5 up to 7.4 sodium phosphate buffer, potassium hydrogen phosphate sodium dihydrogen phosphate buffer as well as citric acid phosphate buffer, potassium sodium glutamate phosphate buffer and succinate buffer.
  • Moisturizing substances such as glycerol, 1,2-propylene glycol, sorbitol, butylene glycol and polyols (for example polyethylene glycol 400) are suitable for the IFN alpha-containing preparation forms according to the invention.
  • Suitable skin-friendly preservatives are e.g. B. p-hydroxybenzoic acid ester (nipa ester), preferably methyl p-hydroxybenzoate; furthermore benzoic acid, sorbic acid, chlorhexidine digluconate, benzalkonium chloride and hexadecyltrimethylammonium bromide (cetrimonium bromide).
  • a suitable penetration promoter compatible with IFN alpha 2 is up to 50% by weight dimethyl sulfoxide (DMSO).
  • Water is ideal as a vehicle for non-alcoholic hydrogels.
  • Suitable pharmaceutical forms for topical application on the skin, in the eye or in the nose or other body cavities are e.g. B. hydrogels. These generally contain 0.1 to 2.0% by weight both of gelatin and of cellulose derivatives and / or polyvinyl alcohols, polyvinylpyrrolidone or polyacrylic acid polymers, the ratios having to be coordinated with one another depending on the desired viscosity. If polyvinyl alcohols or polyvinyl pyrrolidone are used, up to 15% by weight can be required. In the case of the manufacture of vaginal balls, the proportion of gelatin should be increased to 10% by weight, also depending on the desired viscosity.
  • Nipagin M methyl p-hydroxybenzoate
  • Polysorbate 20 polyoxyethylene (20) sorbitan monolaurate (Tween®20);
  • Polysorbate 60 polyoxyethylene (20) sorbitan monostearate (Tween®60),
  • Polysorbate 80 polyoxyethylene (20) sortiban monooleate (Tween®80),
  • the IFN alpha 2 hydrogel is prepared by dissolving Nipagin M in water at 80 ° C. and then cooling the solution to room temperature (RT), the gelatin being introduced into this solution during the cooling phase. After the gelatin has completely dissolved, NaCl, KCL, NaH2PO4 ⁇ 2H2O, K2HPO4 ⁇ 3 H2O, glutamic acid-Na ⁇ H2O and polysorbate 20 are added consecutively at RT to this solution. After subsequent sterile filtration, microbiologically pure hydroxyethyl cellulose is sprinkled in and the solution is heated to 80 ° C. again with stirring.
  • the hydrochloric acid, 1% polysorbate-containing IFN alpha 2 solution consisting of an IFN alpha 2 lyophilisate dissolved in 0.01N HCl, is added with gentle stirring and distributed homogeneously. This mixture is filled under laminar air-flow conditions in sterile aluminum tubes.
  • the IFN-alpha 2 hydrogel is prepared by dissolving benzalkonium chloride in the water at 30 ° C., then adding the propylene glycol at RT and introducing the gelatin into the solution. After the gelatin has completely dissolved, NaCl, Na2HPO4 ⁇ 2H2O, KH2PO4 and polysorbate 80 are added in succession at RT. After sterile filtration has been carried out, microbiologically pure hydroxyethyl cellulose is sprinkled in and the solution is heated to 80 ° C. with stirring for 20 minutes.
  • the hydrochloric acid, polysorbate-containing IFN-alpha-2 solution consisting of an IFN-alpha-2-lyophilisate dissolved in 0.0LN HCl and 0.1% polysorbate 80, is added with careful stirring and homogeneously distributed. This mixture is filled under laminar air flow conditions in sterile aluminum tubes.
  • the IFN-alpha-2 hydrogel is prepared by dissolving chlorhexidine digluconate, glycerol and gelatin at approx. 30 ° C. Then succinic acid is added, adjusted to pH 6.0 with 1N NaOH and polysorbate 20 is added.
  • microbiologically pure hydroxyethyl cellulose is sprinkled in and the solution is heated to 80 ° C. for 20 minutes with stirring. After cooling to RT, the hydrochloric acid, polysorbate-containing IFN-alpha-2 solution is added with gentle stirring and distributed homogeneously. The filling takes place as in Examples 1 and 2.
  • the IFN-alpha-2 hydrogel is prepared by dissolving the hydroxypropylmethyl cellulose in water at 40 ° C. with stirring. The mixture is then cooled to RT, and gelatin is introduced into the solution during the cooling phase. After the solution has been introduced, the following are introduced in succession: benzalkonium chloride, sorbitol solution, citric acid, di-Na hydrogen phosphate, NaCl and polysorbate 60.
  • the hydrochloric acidic, 1% polysorbate-containing IFN-alpha-2 solution is introduced into the finished gel at RT with careful stirring and homogeneously distributed. The filling takes place under laminar air flow conditions in sterile aluminum tubes.
  • the IFN-alpha-2 hydrogel is prepared by stirring the polyacrylic acid and gelatin in water is dissolved at RT, then sorbitol solution, glycerol, NaCl, cetrimonium bromide and polysorbate are added in succession. After the solution is complete, the product is sterile filtered and adjusted to about pH 7.6 with sodium hydroxide solution at RT, during which the gelation occurs. The hydrochloric acid (0.001N), 1% polysorbate-containing IFN-alpha-2 solution is then introduced into this gel at RT with careful stirring and homogeneously distributed. As in the previous examples, the filling takes place under laminar air flow in sterile aluminum tubes.
  • the IFN-alpha-2 ovula is produced by dissolving the hydroxyethyl cellulose together with the gelatin in the glycerol-water mixture, which already contains Na2HPO4 ⁇ 2H2O, KH2PO4 and polysorbate at approx. 40-50 ° C. While still viscous (at approx. 37-40 ° C), the 1% polysorbate-containing, hydrochloric acid (0.00lN) IFN-alpha-2 solution is carefully stirred in and the solution is immediately poured out into suitable, cooled molds for solidification.
  • Fig. 1 to 3 describe the stability curve of an IFN-alpha-2 hydrogel of the above composition without the addition of gelatin, the solid curves with the addition of gelatin filled in aluminum tubes. Each point represents the mean of 6 determinations. The determinations were carried out using the ELISA test.
  • Figure 1 shows the stability curve for minus 10 ° C storage, Figure 2 for plus 4 ° C storage and in Figure 3 for plus 2 ° C storage, in each case comparative.

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Abstract

The pharmaceutical forms are intended for topical administration and employ as vehicle for and stabiliser of the active ingredient a composition which specifically confers special stability on the active ingredient IFN alpha. The forms are essentially composed of hydroxyethylcellulose and gelatin and preferably contain highly purified IFN alpha which has been prepared by DNA recombination as active ingredient.

Description

Abhängig von ihren biologischen und chemischen Eigenschaften und ihrem Syntheseort werden Human-Interferone und Inter­ferone allgemein in drei Gruppen unterteilt, die mit Inter­feron alpha, Interferon beta und Interferon gamma bezeichnet werden entsprechend IFN alpha, IFN beta, IFN gamma. Der Syntheseort von IFN alpha, auf das sich die vorliegende Er­findung bezieht, sind die Leucozyten, die nach Stimulation oder Induktion durch entsprechende Induktoren (z. B. Viren oder poly I· poly C)) IFN alpha synthetisieren und frei­setzen. Mit geeigneten DNA-Sequenzen transformierte Bakte­rien können IFN alpha synthetisieren, die eine dem genuinen Human IFN alpha analoge biologische Aktivität besitzen, mit dem Vorteil, daß hochgereinigte und im Wesentlichen von Kon­taminationen freie Proteine in vergleichsweise großen Mengen erhalten werden können, die für die klinische Anwendung, z.B. gegen verschiedene Virusinfektionen und Malignome, einsetz­bar sind.Depending on their biological and chemical properties and their place of synthesis, human interferons and interferons are generally divided into three groups, which are designated interferon alpha, interferon beta and interferon gamma, corresponding to IFN alpha, IFN beta, IFN gamma. The synthesis site of IFN alpha to which the present invention relates are the leucocytes which, after stimulation or induction by appropriate inducers (e.g. viruses or poly I · poly C)), synthesize and release IFN alpha. Bacteria transformed with suitable DNA sequences can synthesize IFN alpha, which have a biological activity analogous to the real human IFN alpha, with the advantage that highly purified and essentially contaminant-free proteins can be obtained in comparatively large amounts, which are suitable for clinical use , for example against various virus infections and malignancies.

Alle bekannten Interferone wirken antiviral, antiprolifer­ativ und - jedoch verschieden stark - immunmodulierend. An­fänglich als antivirales Agens erkannt, wird heute ihr the­rapeutischer Nutzen auch als antineoplastisches Mittel ge­sehen.All known interferons have an antiviral, antiproliferative and - but different strength - immunomodulating effect. Initially recognized as an antiviral agent, today its therapeutic use is also seen as an antineoplastic agent.

Hinsichtlich Wirkungsqualität und Nebenwirkungsrate kommt der Applikationsroute eine besondere Bedeutung zu. Gerade bei der systemischen Anwendung von Interferonen imponieren die Nachteile, da sie nicht nur das Zielgewebe, sondern auch gesunde, nicht infizierte oder nicht transformierte Zellen oder Organe erreichen und beeinflußen und daher Nebenwir­kungen erzeugen, die einem intravenösen oder intramuskulären Einsatz entgegenstehen können. Außerdem zeigte die systemi­sche Applikation nicht immer die erwünschte Effektivität. In einzelnen Fällen mag eine direkte Injektion zwar möglich sein, das Mittel der Wahl, z. B. bei Virus - oder tumorbe­dingten malignen oder anderen mit Interferon therapierbaren Erkrankungen, die einer äußerlichen und lokalen Behandlung zugänglich sind (z. B. Haut, Augen, Nase), ist aber eher die topische Anwendung von Interferon bzw von IFN alpha, auf das sich diese Erfindung bezieht. In jüngster Zeit wurde von verschiedenen Seiten über die topische Applikation von Human-Leucocyten-Interferon, synonym mit IFN alpha, berich­tet, wobei überraschenderweise eine hohe Erfolgsquote bei verschiedenen malignen Erkrankungen festgestellt werden konnte (z. B. Ikic et al, Lancet, l022-24, l98l, Vol.l ; Ikic et al., Lancet l025-27, l98l, Vol.l). Es ist abzusehen, daß der topischen Anwendung von Interferon alpha zur Behand­lung verschiedener viraler oder tumorbedingter Erkrankungen in Zukunft ein zunehmender medizinischer Stellenwert zukom­men wird.The application route is of particular importance with regard to the quality of effects and the rate of side effects. The disadvantages are particularly impressive when it comes to the systemic use of interferons, since they reach and influence not only the target tissue, but also healthy, uninfected or untransformed cells or organs and therefore produce side effects that can prevent intravenous or intramuscular use. In addition, the systemic application did not always show the desired effectiveness. In individual cases, a direct injection may be possible, the agent of choice, e.g. B. for virus - or tumor - related malignancies or other diseases which can be treated with interferon and which are accessible to external and local treatment (e.g. skin, eyes, nose), but is rather the topical application of interferon or IFN alpha, on which this invention relates. The topical application of human leucocyte interferon, synonymous with IFN alpha, has recently been reported by various parties, with a surprisingly high success rate in various malignant diseases being found (e.g. Ikic et al, Lancet, l022- 24, l98l, vol. 1; Ikic et al., Lancet l025-27, l98l, vol. L). It is foreseeable that the topical use of interferon alpha for the treatment of various viral or tumor-related diseases will become increasingly important in the medical field.

Mit der topischen Anwendung von IFN alpha als auch allgemein von Interferonen sind unter verschiedenen Gesichtspunkten erhebliche Schwierigkeiten verbunden. Unter dem Gesichts­punkt der pharmazeutischen Formulierung ist zu fordern, daß das biologisch aktive IFN alpha eine genügend hohe Stabili­tät erhält, um über eine ausreichend lange Zeit sowohl bei Raumtemperatur als auch bei Körpertemperatur zu wirken bzw. über einen geeigneten Zeitraum von mehreren Stunden "in situ" wirksam zu bleiben. Bekannt ist, daß IFN alpha ein sehr temperaturinstabiles Protein ist. Erhebliche Aktivi­tätsverluste von bis zu 80% wurden innerhalb von wenigen Wochen bei 4° C bei einem Human-Leucocyten-Interferon-halti­gen Gel beobachtet, das als Gelkomponente Carboxymethylcel­lulose enthielt (Moller B.R. et al., Obstetrics & Gynecology 62, 625-629, l983) und für die topische Anwendung zur Be­handlung des Carcinoma in situ der Cervix eingesetzt wurde.The topical application of IFN alpha and interferons in general is associated with considerable difficulties from various points of view. From the point of view of the pharmaceutical formulation, it must be demanded that the biologically active IFN alpha obtains a sufficiently high stability to act both at room temperature and at body temperature for a sufficiently long time or over a suitable period of several hours "in situ" to remain effective. It is known that IFN alpha a very temperature-unstable protein. Significant activity losses of up to 80% were observed within a few weeks at 4 ° C. on a gel containing human leucocyte interferon, which contained carboxymethyl cellulose as the gel component (Moller BR et al., Obstetrics & Gynecology 62 , 625-629, 1983) ) and the cervix was used for topical application for the treatment of carcinoma in situ.

Die daher notwendigen stabilitätserhaltenden Komponenten müssen so beschaffen sein, daß sie weder Unverträglichkeits­reaktionen am Ort der Applikation verursachen, denkbar durch Wirkung von biologisch aktiven Fremdsubstanzen, noch die Pe­netration von IFN alpha beeinträchtigen oder die Diffusion verhindern.The necessary stability-maintaining components must be such that they neither cause intolerance reactions at the application site, conceivable through the action of biologically active foreign substances, nor impair the penetration of IFN alpha or prevent diffusion.

In der letzten Zeit wurden verschiedene Komponenten zur Sta­bilisierung von Interferon benutzt, wobei Estis, Evans und Testa Hydroxyäthylcellulose als Trägermaterial zur Herstel­lung von Interferon-haltigen Gelen oder Salben eingesetzt haben, aber erhebliche Aktivitätsverluste von IFN alpha, IFN beta oder IFN gamma unter anwendungsrealistischen Bedin­gungen nur durch Zusatz eines Proteaseinhibitors verhindern konnten (EP-A-0 l42 345).Various components have recently been used to stabilize interferon, with Estis, Evans and Testa using hydroxyethyl cellulose as the carrier material for the production of gels or ointments containing interferon, but only significant losses in activity of IFN alpha, IFN beta or IFN gamma under realistic application conditions could be prevented by adding a protease inhibitor (EP-A-0 142 425).

Dem Fachmann ist bekannt, daß mit Hydroxyäthylcellulose ein zur Herstellung von Gelen dieser oder auch anderer Art best­geeignetes Mittel, z. B. aufgrund seiner Hitzestabilität, vorliegt und kommerziell erhältlich ist.It is known to the person skilled in the art that hydroxyethyl cellulose is a most suitable agent for the production of gels of this or other type, e.g. B. because of its heat stability, is present and is commercially available.

Der besondere Vorteil von Hydroxyäthylcellulose zur Herstel­lung einer IFN alpha-haltigen Matrix liegt gegenüber anderen Trägern darin, daß sie bei Siedetemperatur von Wasser nicht koaguliert bzw. geliert, gut wasserlöslich ist, und daß die Viskosität einer solchen Lösung mit abnehmender Temperatur zunimmt.The particular advantage of hydroxyethyl cellulose for the production of an IFN alpha-containing matrix compared to other carriers is that it does not coagulate or gel at the boiling point of water, is readily water-soluble, and that the viscosity of such a solution increases with decreasing temperature.

Eine Stabilisierung von Interferon bzw. von IFN alpha durch Hydroxyäthylcellulose allein und unter physiologi­schen Temperaturen ist nicht zu erreichen. Auch ist dem Fachmann bekannt, daß nicht hochgereinigte und daher kon­taminierte Proteinpräparate - wie z.B. Interferone - unter geeigneten Bedingungen der Hydrolyse durch Proteasen ausge­setzt sind. Andere Versuche, in pharmazeutischen Präparaten enthaltene Interferone eine geeignete Stabilität zu ver­leihen, wurden von Akagi, Miura und Hoshino (EP-A-0 l50 067) mit von Hydroxyäthylcellulose chemisch unterschiedlichen Polysacchariden, wie Dextran und Hydroxyäthylstärke unter Zusatz von Human Serum Albumin, von Mono- oder Disacchariden oder von Aminosäuren durchgeführt, wobei Präparate vornehm­lich für die parenterale, systemische Anwendungsroute via intravenöser oder intramuskulärer Injektion beschrieben wur­den. Darüber hinaus wurde von Terano nur allein auf Basis einer chemisch modifizierten Gelatine, ebenfalls für ein zu injizierendes pharmazeutisches Interferon-Präparat, eine Stabilisierung von Interferon in wässriger Lösung beschrie­ben, um damit Human Serum Albumin als bekanntes Interferon­stabilisierendes Mittel für die Injektion zu substituieren (EP-A-0 l62 332). Es ist dem Fachmann seit langem bekannt, daß Gelatine per se einen auf Interferone stabilisierenden Effekt aufweist (Sedmak J.J. and Grossberg, S.E. Tex. Rep. Biol. Med. 4l, 274-279, l982), und daß Gelatine und chemisch modifizierte Derivate davon auf einige andere biologische Proteine einen gewissen stabilisierenden Effekt ausüben (z.B. DE-A-l ll8 732, EP-A-0 l56 242).Stabilization of interferon or IFN alpha by hydroxyethyl cellulose alone and under physiological temperatures cannot be achieved. It is also known to the person skilled in the art that protein preparations which are not highly purified and therefore contaminated - such as e.g. Interferons - are exposed to hydrolysis by proteases under suitable conditions. Other attempts to give interferons contained in pharmaceutical preparations a suitable stability have been made by Akagi, Miura and Hoshino (EP-A-0 150 067) with polysaccharides chemically different from hydroxyethyl cellulose, such as dextran and hydroxyethyl starch with the addition of human serum albumin, by Mono - or disaccharides or of amino acids, preparations being described primarily for the parenteral, systemic route of use via intravenous or intramuscular injection. In addition, Terano has described stabilization of interferon in aqueous solution only on the basis of a chemically modified gelatin, also for a pharmaceutical interferon preparation to be injected, in order to substitute human serum albumin as a known interferon-stabilizing agent for injection (EP- A-0 l62 332). It has long been known to the person skilled in the art that gelatin per se has a stabilizing effect on interferons (Sedmak JJ and Grossberg, SE Tex. Rep. Biol. Med. 4l, 274-279, l982), and that gelatin and chemically modified derivatives thereof exert a certain stabilizing effect on some other biological proteins (eg DE-Al ll8 732, EP-A-0 l56 242).

Für die Stabilisierung von Interferon in Gelen, Salben, Sup­positorien, Sprays usw. zur topischen Anwendung sind auch verschiedene Zuckeralkohole wie Glycerol, Erythoritol, Ara­bitol, Xylitol, Sorbitol und Mannitol sowie verschiedene Zuckersäuren wie Iduronsäure, Galacturonsäure und andere Uronsäuren,Ascorbinsäure und deren Salze als auch verschie­dene organische Puffersysteme wie Citratpuffer, Succinat­ puffer, Tartratpufer, Fumaratpuffer, Glyconatpuffer, Oxalat­puffer, Lactatpuffer und Acetatpuffer offenbart worden, in Verbindung mit verschiedenen pharmazeutischen Trägerstoffen wie Wachse, Natriumcarboxymethylcellulose, Carboxyvinylderi­vate und Wasser (EP-A-0 080 879).For the stabilization of interferon in gels, ointments, suppositories, sprays etc. for topical use, there are also various sugar alcohols such as glycerol, erythoritol, arabitol, xylitol, sorbitol and mannitol as well as various sugar acids such as iduronic acid, galacturonic acid and other uronic acids, ascorbic acid and their salts also various organic buffer systems such as citrate buffer, succinate Buffer, tartrate buffer, fumarate buffer, glyconate buffer, oxalate buffer, lactate buffer and acetate buffer have been disclosed in connection with various pharmaceutical carriers such as waxes, sodium carboxymethyl cellulose, carboxyvinyl derivatives and water (EP-A-0 080 879).

Diese letztgenannten als auch die vorhergenannten Inter­feron-stabilisierenden Komponenten sowie vor allem die Zu­sammensetzungen der pharmazeutischen Formulierungen und deren Anwendungsmodalitäten (z.B. EP-A-0 l62 332) sind grundsätzlich verschieden von der vorliegenden Erfindung und mit dieser nicht vergleichbar.These latter as well as the aforementioned interferon-stabilizing components and especially the compositions of the pharmaceutical formulations and their application modalities (e.g. EP-A-0 l62 332) are fundamentally different from the present invention and cannot be compared with it.

Die Aufgabe der vorliegenden Erfindung war es, eine Träger­matrix für hochgereinigtes Interferon alpha, speziell für rekombinant hergestelltes IFN alpha zu finden, die mindes­tens den obig aufgezählten Anforderungen entsprechen und zum anderen eine sowohl unter herstellungstechnischen als auch unter ökonomischen Gesichtspunkten so günstig wie mögliche, d.h. einfache Zusammensetzung, aufweisen sollte, ohne eine Einbuße einer geforderten optimalen Wirksamkeit des in der Trägermatrix enthaltenen biologisch aktiven Wirkstoffes IFN alpha zur topischen Anwendung zu bewirken. Durch die vorliegende Erfindung wurde dieser Aufgabenkomplex dadurch gelöst, daß Hydroxyäthylcellulose und Gelatine in bestimmten Mischungsverhältnissen miteinander vereint wurden und somit ein Träger und Stabilisator für IFN alpha geschaffen werden konnte, der die oben beschriebenen Anforderungen nicht nur erfüllt, sondern überraschenderweise dem IFN alpha eine be­sonders stabile biologische Aktivität verleiht und somit be­stens für die topische Anwendung von hochgereinigtem insbe­sondere über rekombinante DNA-Technologie hergestelltem IFN alpha geeignet ist.The object of the present invention was to find a carrier matrix for highly purified interferon alpha, especially for recombinantly produced IFN alpha, which at least meet the requirements enumerated above and, on the other hand, is as cheap as possible both from a manufacturing and economic point of view, i.e. simple composition, should have, without a loss of a required optimal effectiveness of the biologically active active ingredient IFN alpha contained in the carrier matrix for topical application. The present invention achieved this complex of problems by combining hydroxyethyl cellulose and gelatin in certain mixing ratios and thus creating a carrier and stabilizer for IFN alpha which not only meets the requirements described above, but surprisingly also makes IFN alpha particularly stable gives biological activity and is therefore ideally suited for the topical application of highly purified IFN alpha, in particular produced using recombinant DNA technology.

Ein weiterer Vorteil dieser Erfindung besteht darin, daß der gefundene Träger/Stabilisator-Komplex frei von Human Serum Albumin ist, das bekannterweise und insbesondere bei der to­pischen Anwendung gerade über längere Zeiträume zu lokalen Immunreaktionen bis zu Allergosen führen kann. Die vorlie­gende Erfindung bezieht sich daher auf eine verbesserte pharmazeutische Formulierung für die topische Anwendung von IFN alpha. Erfindungsgemäß wurde dabei festgestellt, daß beide als Träger und Stabilisator fungierenden Einzelkom­ponenten zusammen, nämlich Hydroxyäthylcellulose und Gela­tine, eine über das zu erwartende Maß hinausgehende aktivi­tätserhaltende Wirkung auf IFN alpha ausüben, wobei gerade Berücksichtigung finden muß, daß IFN alpha und Interferone allgemein dafür bekannt sind, z.B. gegenüber physikalischen Einflüssen, instabile Proteine zu sein - sowohl in "nackter" Form als auch inkorporiert in anderen Gelmatrizes (z. B. Moller B.R. et al., siehe oben), so daß den erfindungsge­mäßen Zubereitungsformen überraschend und in unvorherseh­barer Weise ein synergistischer Effekt hinsichtlich der Sta­bilistätserhöhung von IFN alpha eigen bzw. inhärent ist. So­mit dient die Gelatine in der vorliegenden Erfindung nicht für sich allein als stabilisierendes Mittel, sondern ent­faltet erst im Komplex zusammen mit Hydroxyäthylcellulose den festgestellten und erstaunlichen synergistischen Stabi­lisierungseffekt für IFN alpha, auf den sich diese Erfindung bezieht.Another advantage of this invention is that the carrier / stabilizer complex found is free of human serum Albumin is, which is known to lead to local immune reactions and allergies, especially when used topically, especially over longer periods of time. The present invention therefore relates to an improved pharmaceutical formulation for the topical application of IFN alpha. According to the invention, it was found that the two individual components acting as carrier and stabilizer, namely hydroxyethyl cellulose and gelatin, exert an activity-preserving effect on IFN alpha which goes beyond what is to be expected, taking into account the fact that IFN alpha and interferons are generally known for eg against physical influences to be unstable proteins - both in "naked" form and incorporated in other gel matrices (e.g. Moller BR et al., see above), so that the preparation forms according to the invention surprisingly and in an unpredictable manner have a synergistic effect is inherent or inherent in increasing the stability of IFN alpha. Thus, the gelatin in the present invention does not in itself serve as a stabilizing agent, but only develops the established and astonishing synergistic stabilizing effect for IFN alpha to which this invention relates in combination with hydroxyethyl cellulose.

Die erfindungsgemäßen, topisch zu applizierenden pharmazeu­tischen Zubereitungsformen für IFN alpha werden vorzugsweise als Virusstatika angewendet, wobei der therapeutische Ein­satz im dermalen, nasalen, ophthalmischen und intravaginalen Bereich, z.B. bei Herpes labialis, Herpes genitalis inclusive Papilloma-Infektionen, Warzen und bei Herpes Zoster-Infek­tionen, erfolgt. Der Anwendungsumfang umfaßt auch die to­pische Behandlung von Paracancerosen und Cancerosen.The pharmaceutical preparation forms for IFN alpha according to the invention, which are to be applied topically, are preferably used as virus static agents, the therapeutic use in the dermal, nasal, ophthalmic and intravaginal area, for example for herpes labialis, genital herpes including papilloma infections, warts and herpes zoster infections , he follows. The scope of application also includes the topical treatment of paracanceroses and canceroses.

Gegenstand dieser Erfindung sind topisch anzuwendende phar­mazeutische Zubereitungsformen mit einer Stabilisierung des darin enthaltenen Wirkstoffs Human Interferon alpha (IFN alpha), bestehend aus l. einem Träger und Stabilisator im Komplex für den Wirkstoff, 2. einem Antiadhäsionsmittel und 3. gegebenfalls sonstigen üblichen Wirkstoffen wie Puf­fer und Salze, Feuchthalter, Konservierungsstoffe, Penetra­tionsförderer, Vehikel.This invention relates to topical pharmaceutical preparation forms with a stabilization of the active ingredient contained therein, human interferon alpha (IFN alpha), consisting of l. a carrier and stabilizer in the complex for the active ingredient, 2. an antiadhesive and 3. if appropriate, other customary active ingredients such as buffers and salts, humectants, preservatives, penetration promoters, vehicles.

Als Träger und Stabilisator für IFN alpha werden bekannte Stoffe wie Cellulosederivate (Carboxymethylcellulose, Methylcellulose, Hydroxypropylmethylcellulose) sowie Poly­vinylalkohole, Polyvinylpyrrolidon, Polyacrylsäure-Polymeri­sate (Carbopol® und Mischungen davon zusammen mit Gela­tine, sauer oder alkalisch aufgeschlossen, eingesetzt. Als bevorzugter Träger und Stabilisator für IFN alpha wird Hy­droxyäthylcellulose mit einem Substitutionsgrad von 2,5 in bestimmten Mischungsverhältnissen von jeweils 0,l bis 2 Gew.% und Gelatine von 0,l bis l0 Gew.%, jeweils bezogen auf das fertige Produkt, benutzt. Verwendet man Polyvinylalko­hole sowie Polyvinylpyrrolidon, so werden bis l5 Gew.% hier­von eingesetzt. Damit wird eine besondere Stabilisierung speziell von IFN alpha erreicht, wie es auch die durchge­führten Versuche über das Stabilitätsverhalten beispielhaft belegen und wie es die in Abb. l bis 3 gezeigten Kurvenver­läufe, die einen Versuchszeitraum von l4 Wochen bei Lager­temparaturen jeweils von -l0, +4 und +2l°C umfassen, bei­spielhaft zeigen.Known substances such as cellulose derivatives (carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose) as well as polyvinyl alcohols, polyvinyl pyrrolidone, polyacrylic acid polymers (Carbopol® and mixtures thereof together with gelatin, acid or alkaline digested) are used as carriers and stabilizers for IFN alpha. Preferred carriers and stabilizers for IFN alpha uses hydroxyethyl cellulose with a degree of substitution of 2.5 in certain mixing ratios of 0.1 to 2% by weight and gelatin of 0.1 to 10% by weight, based in each case on the finished product, using polyvinyl alcohols and polyvinyl pyrrolidone , up to 15% by weight of this is used, thus achieving a special stabilization especially of IFN alpha, as is also demonstrated by the tests carried out on the stability behavior and how the curves shown in Fig. 1 to 3, which have a test period of 14 weeks for storage repairs each include from -l0, +4 and + 2l ° C, show an example.

Als Antiadhäsionsmittel zur besseren Spreitung der Gele und als antiadhäsive Filtrationshilfe können anionaktive, ka­tionaktive sowie neutrale oberflächenaktive Substanzen be­nutzt werden, vorzugsweise werden polyäthoxylierte Sorbi­tan-Ester wie Polysorbat 20, 60, 80 benutzt.Als anorganische und organische Pufferlösungen haben sich mit einem für IFN alpha - Präparationen optimalen pH im Bereich von 6,5 bis 7,4 Natriumphosphat-Puffer, Kaliumhydrogenphosphat-Na­triumdihydrogenphosphat-Puffer sowie Citronensäurephos­phat-Puffer, Kalium-Natrium-glutamat-phosphat-Puffer und Succinat-Puffer bewährt.Anionic, cationic and neutral surface-active substances can be used as anti-adhesive agents for better spreading of the gels and as anti-adhesive filtration aid, preferably polyethoxylated sorbitan esters such as polysorbate 20, 60, 80 are used. As inorganic and organic buffer solutions with an IFN alpha - Preparations optimal pH in the range of 6.5 up to 7.4 sodium phosphate buffer, potassium hydrogen phosphate sodium dihydrogen phosphate buffer as well as citric acid phosphate buffer, potassium sodium glutamate phosphate buffer and succinate buffer.

Für die erfindungsgemäßen IFN alpha haltigen Zubereitungs­formen eignen sich Feuchthaltesubstanzen wie Glycerin, l,2-Propylenglykol, Sorbit, Butylengklykol sowie Polyole (z. B. Polyethylenglykol 400).Moisturizing substances such as glycerol, 1,2-propylene glycol, sorbitol, butylene glycol and polyols (for example polyethylene glycol 400) are suitable for the IFN alpha-containing preparation forms according to the invention.

Geeignete hautverträgliche Konservierungsstoffe sind z. B. p-Hydroxybenzoesäureester (Nipa-Ester), vorzugsweise p-Hy­droxybenzoesäuremethylester; des weiteren Benzoesäure, Sor­binsäure, Chlorhexidindigluconat, Benzalkoniumchlorid und Hexadecyltrimethylammoniumbromid (Cetrimoniumbromid).Suitable skin-friendly preservatives are e.g. B. p-hydroxybenzoic acid ester (nipa ester), preferably methyl p-hydroxybenzoate; furthermore benzoic acid, sorbic acid, chlorhexidine digluconate, benzalkonium chloride and hexadecyltrimethylammonium bromide (cetrimonium bromide).

Ein geeigneter mit IFN alpha 2 kompatibler Penetrationsför­derer ist bis zu 50 Gew.% Dimethylsulfoxid (DMSO).A suitable penetration promoter compatible with IFN alpha 2 is up to 50% by weight dimethyl sulfoxide (DMSO).

Als Vehikel ist bei nichtalkoholischen Hydrogelen Wasser be­stens geeignet.Water is ideal as a vehicle for non-alcoholic hydrogels.

Geeignete pharmazeutische Formen für die topische Anwendung auf der Haut, im Auge oder in der Nase oder sonstigen Kör­perhöhlen sind z. B. Hydrogele. Diese enthalten im allgemei­nen 0,l bis 2,0 Gew.% sowohl an Gelatine als auch an Cellu­losederivaten und/oder Polyvinylalkohole, Polyvinylpyrroli­don oder Polyacrylsäure - Polymerisate, wobei die Verhält­nisse je nach gewünschter Viskosität aufeinander abgestimmt werden müssen. Verwendet man Polyvinylalkohole oder Poly­vinylpyrrolidon, so können hiervon bis zu l5 Gew.% erforder­lich werden. Im Falle der Fertigung von Vaginalkugeln ist der Anteil an Gelatine bis auf l0 Gew.% zu erhöhen, eben­falls in Abhängigkeit von der gewünschten Viskosität.Suitable pharmaceutical forms for topical application on the skin, in the eye or in the nose or other body cavities are e.g. B. hydrogels. These generally contain 0.1 to 2.0% by weight both of gelatin and of cellulose derivatives and / or polyvinyl alcohols, polyvinylpyrrolidone or polyacrylic acid polymers, the ratios having to be coordinated with one another depending on the desired viscosity. If polyvinyl alcohols or polyvinyl pyrrolidone are used, up to 15% by weight can be required. In the case of the manufacture of vaginal balls, the proportion of gelatin should be increased to 10% by weight, also depending on the desired viscosity.

In den folgenden Beispielen wird die Herstellung von topisch anzuwendenden pharmazeutischen Zubereitungsformen zur Stabi­lisierung des darin enthaltenen IFN alpha im Detail be­schrieben, die sich gemäß der vorliegenden Erfindung in der Hauptsache aus Celluloseerivaten und Gelatine, Antiadhäs­ionsmitteln sowie dem darin enthaltenen Wirkstoff IFN alpha und verschiedenen Zusatzstoffen zusammensetzen. Es wird je­doch vorausgesetzt, daß viele Modifikationen und Verände­rungen durchführbar sind und den Gegenstand der Erfindung einschließen, ohne sich von dem dieser Erfindung zugrunde­liegenden Umfang zu entfernen, und daß die aufgeführten Bei­spiele die Erfindung nicht einschränken.
In den Beispielen bedeuten:

Nipagin M = p-Hydroxybenzoesäuremethylester,
Polysorbat 20 = Polyoxyethylen(20)sorbitanmonolaurat (Tween®20);
Polysorbat 60 = Polyoxyethylen(20)sorbitanmonostearat (Tween®60),
Polysorbat 80 = Polyoxyethylen(20)sortibanmonooleat (Tween®80),
Benzalkoniumchlorid = Mischung aus Alkyldimethylbenzylammonium­chloriden mit Alkylgruppen von 8 bis l8 Kohlenstoffatomen,
Cetrimoniumbromid = Hexadecyltrimethylammoniumbromid,
3,2 × l0⁸ I.E. Interferon-alpha-2-arg entsprechen l mg dieser Substanz.

Figure imgb0001
The following examples describe in detail the preparation of topical pharmaceutical preparation forms for stabilizing the IFN alpha contained therein, which according to the present invention mainly consist of cellulose derivatives and gelatin, anti-adhesive agents and the active ingredient IFN alpha contained therein and various additives. However, it is anticipated that many modifications and changes may be made without departing from the scope of the invention, and that the examples given do not limit the invention.
In the examples:

Nipagin M = methyl p-hydroxybenzoate,
Polysorbate 20 = polyoxyethylene (20) sorbitan monolaurate (Tween®20);
Polysorbate 60 = polyoxyethylene (20) sorbitan monostearate (Tween®60),
Polysorbate 80 = polyoxyethylene (20) sortiban monooleate (Tween®80),
Benzalkonium chloride = mixture of alkyldimethylbenzylammonium chlorides with alkyl groups of 8 to 18 carbon atoms,
Cetrimonium bromide = hexadecyltrimethylammonium bromide,
3.2 × 10 IU of interferon-alpha-2-arg correspond to 1 mg of this substance.

Figure imgb0001

Die Präparation des IFN alpha 2 - Hydrogels erfolgt da­durch, daß Nipagin M in 80°C heißem Wasser gelöst und die Lösung anschließend auf Raumtemperatur (RT) abgekühlt wird, wobei während der Abkühlphase die Gelatine in diese Lösung eingebracht wird. Nach der vollständigen Lösung der Gelatine werden bei RT konsekutiv NaCl, KCL, NaH₂PO₄ × 2H₂O, K₂HPO₄ × 3 H₂O, Glutaminsäure-Na × H₂O sowie Polysor­bat 20 zu dieser Lösung dazugegeben. Nach anschließender Sterilfiltration wird mikrobiologisch reine Hydroxyäthylcel­lulose eingestreut und die Lösung unter Rühren wieder auf 80°C erwärmt. Nach dem Abkühlen auf RT wird unter vorsichti­gem Rühren die salzsaure, l % Polysorbat-haltige IFN alpha 2 - Lösung, bestehend aus einem in 0.0l N HCl gelöstem IFN alpha 2 - Lyophilisat, dazugegeben und homogen verteilt. Die Abfüllung dieses Gemisches erfolgt unter laminaren Air-flow-­Bedingungen in sterile Aluminium-Tuben.

Figure imgb0002
The IFN alpha 2 hydrogel is prepared by dissolving Nipagin M in water at 80 ° C. and then cooling the solution to room temperature (RT), the gelatin being introduced into this solution during the cooling phase. After the gelatin has completely dissolved, NaCl, KCL, NaH₂PO₄ × 2H₂O, K₂HPO₄ × 3 H₂O, glutamic acid-Na × H₂O and polysorbate 20 are added consecutively at RT to this solution. After subsequent sterile filtration, microbiologically pure hydroxyethyl cellulose is sprinkled in and the solution is heated to 80 ° C. again with stirring. After cooling to RT, the hydrochloric acid, 1% polysorbate-containing IFN alpha 2 solution, consisting of an IFN alpha 2 lyophilisate dissolved in 0.01N HCl, is added with gentle stirring and distributed homogeneously. This mixture is filled under laminar air-flow conditions in sterile aluminum tubes.
Figure imgb0002

Die Herstellung des IFN-alpha 2-Hydrogels erfolgt da­durch, daß Benzalkoniumchlorid bei 30° C in dem Wasser gelöst wird, anschließend wird bei RT das Propylenglykol zugesetzt und die Gelatine in die Lösung eingebracht. Nach vollständiger Lösung der Gelatine werden bei RT nacheinander NaCl, Na₂HPO₄ × 2H₂O, KH₂PO₄ sowie Polysorbat 80 dazugegeben. Nach erfolgter Sterilfiltra­tion wird mikrobiologisch reine Hydroxyäthylcellulose eingestreut und die Lösung unter Rühren 20 Min. auf 80° C erwärmt. Nach dem Abkühlen auf RT wird unter vorsichtigem Rühren die salzsaure, polysorbathaltige IFN-alpha-2-Lö­sung, bestehend aus einem in 0,0lN HCl und 0,l % Polysor­bat 80 gelöstem IFN-alpha-2-Lyophilisat, dazugegeben und homogen verteilt. Die Abfüllung dieses Gemisches erfolgt unter laminaren Air Flow-Bedingungen in sterilen Alumini­umtuben.

Figure imgb0003
The IFN-alpha 2 hydrogel is prepared by dissolving benzalkonium chloride in the water at 30 ° C., then adding the propylene glycol at RT and introducing the gelatin into the solution. After the gelatin has completely dissolved, NaCl, Na₂HPO₄ × 2H₂O, KH₂PO₄ and polysorbate 80 are added in succession at RT. After sterile filtration has been carried out, microbiologically pure hydroxyethyl cellulose is sprinkled in and the solution is heated to 80 ° C. with stirring for 20 minutes. After cooling to RT, the hydrochloric acid, polysorbate-containing IFN-alpha-2 solution, consisting of an IFN-alpha-2-lyophilisate dissolved in 0.0LN HCl and 0.1% polysorbate 80, is added with careful stirring and homogeneously distributed. This mixture is filled under laminar air flow conditions in sterile aluminum tubes.
Figure imgb0003

Die Herstellung des IFN-alpha-2-Hydrogels erfolgt da­durch, daß Chlorhexidindigluconat, Glycerin und Gelatine bei ca. 30° C in Lösung gebracht werden. Anschließend wird Bernsteinsäure zugesetzt, mit lN NaOH auf pH 6,0 eingestellt und Polysorbat 20 zugegeben.The IFN-alpha-2 hydrogel is prepared by dissolving chlorhexidine digluconate, glycerol and gelatin at approx. 30 ° C. Then succinic acid is added, adjusted to pH 6.0 with 1N NaOH and polysorbate 20 is added.

Dann wird mikrobiologisch reine Hydroxyäthylcellulose eingestreut und die Lösung unter Rühren 20 Min. auf 80° C erwärmt. Nach dem Abkühlen auf RT wird unter vorsichtigem Rühren die salzsaure, polysorbathaltige IFN-alpha-2-Lö­sung dazugegeben und homogen verteilt. Die Abfüllung er­folgt wie bei Beispiel l und 2.

Figure imgb0004
Then microbiologically pure hydroxyethyl cellulose is sprinkled in and the solution is heated to 80 ° C. for 20 minutes with stirring. After cooling to RT, the hydrochloric acid, polysorbate-containing IFN-alpha-2 solution is added with gentle stirring and distributed homogeneously. The filling takes place as in Examples 1 and 2.
Figure imgb0004

Die Herstellung des IFN-alpha-2-Hydrogels erfolgt da­durch, daß in 40° C warmem Wasser die Hydroxypropyl­methylcellulose unter Rühren gelöst wird. Anschließend wird auf RT abgekühlt, während der Abkühlphase wird Gela­tine in die Lösung eingebracht. Nach erfolgter Lösung werden nacheinander eingetragen: Benzalkoniumchlorid, Sorbitlösung, Citronensäure, Di-Na-Hydrogenphosphat, NaCl und Polysorbat 60. In das fertige Gel wird bei RT unter vorsichtigem Rühren die salzsaure, l % Polysorbat-haltige IFN-alpha-2-Lösung eingebracht und homogen verteilt.Die Abfüllung erfolgt unter Laminar-Air-Flow-Bedingungen in sterile Aluminium-Tuben.

Figure imgb0005
The IFN-alpha-2 hydrogel is prepared by dissolving the hydroxypropylmethyl cellulose in water at 40 ° C. with stirring. The mixture is then cooled to RT, and gelatin is introduced into the solution during the cooling phase. After the solution has been introduced, the following are introduced in succession: benzalkonium chloride, sorbitol solution, citric acid, di-Na hydrogen phosphate, NaCl and polysorbate 60. The hydrochloric acidic, 1% polysorbate-containing IFN-alpha-2 solution is introduced into the finished gel at RT with careful stirring and homogeneously distributed.The filling takes place under laminar air flow conditions in sterile aluminum tubes.
Figure imgb0005

Die Herstellung des IFN-alpha-2-Hydrogels erfolgt dadurch, daß die Polyacrylsäure und Gelatine in Wasser unter Rühren bei RT gelöst wird, anschließend werden nacheinander Sor­bitlösung, Glycerin, NaCl, Cetrimoniumbromid und Polysor­bat eingetragen. Nach vollständiger Lösung wird steril filtriert und unter Rühren bei RT auf ca. pH 7,6 mit Natronlauge eingestellt, dabei tritt die Gelierung ein. In dieses Gel wird bei RT dann unter vorsichtigem Rühren die salzsaure (0,00lN), l % Polysorbat-haltige IFN-alpha-­2-Lösung eingebracht und homogen verteilt. Die Abfüllung erfolgt wie in den bisherigen Beispielen unter Laminar-­Air-Flow in sterile Aluminium-Tuben.

Figure imgb0006
The IFN-alpha-2 hydrogel is prepared by stirring the polyacrylic acid and gelatin in water is dissolved at RT, then sorbitol solution, glycerol, NaCl, cetrimonium bromide and polysorbate are added in succession. After the solution is complete, the product is sterile filtered and adjusted to about pH 7.6 with sodium hydroxide solution at RT, during which the gelation occurs. The hydrochloric acid (0.001N), 1% polysorbate-containing IFN-alpha-2 solution is then introduced into this gel at RT with careful stirring and homogeneously distributed. As in the previous examples, the filling takes place under laminar air flow in sterile aluminum tubes.
Figure imgb0006

Die Herstellung der IFN-alpha-2-Ovula erfolgt dadurch, daß die Hydroxyäthylcellulose zusammen mit der Gelatine im Glycerin-Wassergemisch, welches schon Na₂HPO₄ × 2H₂O, KH₂PO₄ sowie Polysorbat enthält bei ca. 40-50°C gelöst wird. Im noch viskosen Zustand (bei ca. 37-40°C) wird die l % Polysorbat-haltige, salzsaure (0,00lN) IFN-alpha-2-Lösung vorsichtig eingerührt und die Lösung sofort zur Erstarrung in geeignete, gekühlte Formen ausgegossen.The IFN-alpha-2 ovula is produced by dissolving the hydroxyethyl cellulose together with the gelatin in the glycerol-water mixture, which already contains Na₂HPO₄ × 2H₂O, KH₂PO₄ and polysorbate at approx. 40-50 ° C. While still viscous (at approx. 37-40 ° C), the 1% polysorbate-containing, hydrochloric acid (0.00lN) IFN-alpha-2 solution is carefully stirred in and the solution is immediately poured out into suitable, cooled molds for solidification.

Beispiel 7 (Stabilitätstests, Hydrogel ohne Gelatine-vs. mit Gelatine-Zusatz) Example 7 (stability tests, hydrogel without gelatin vs. with gelatin addition)

Figure imgb0007
Figure imgb0007

Die punktierten Kurven in Abb.l bis 3 beschreiben den Sta­bilitätsverlauf eines IFN-alpha-2-Hydrogeles obiger Zusam­mensetzung ohne Gelatine-Zusatz, die durchgezogenen Kurven mit Gelatine-Zusatz abgefüllt in Aluminium-Tuben. Jeder Punkt stellt den Mittelwert aus 6 Bestimmungen dar. Die Be­stimmungen wurden mit dem ELISA-Test durchgeführt.The dotted curves in Fig. 1 to 3 describe the stability curve of an IFN-alpha-2 hydrogel of the above composition without the addition of gelatin, the solid curves with the addition of gelatin filled in aluminum tubes. Each point represents the mean of 6 determinations. The determinations were carried out using the ELISA test.

In Abbildung l ist der Stabilitätsverlauf bei Minus l0°C Lagerung, in Abbildung 2 bei Plus 4°C Lagerung und in Ab­bildung 3 bei Plus 2l°C Lagerung, jeweils vergleichend, wiedergegeben. Figure 1 shows the stability curve for minus 10 ° C storage, Figure 2 for plus 4 ° C storage and in Figure 3 for plus 2 ° C storage, in each case comparative.

Das Stabilitätsverhalten wurde über einen Zeitraum von l4 Wochen (= 3 l/2 Monate) verfolgt. Dabei konnte erstaunlich­erweise und völlig unerwartet festgestellt werden, daß mit zunehmender Lagertemperatur die Stabilitätsunterschiede deutlicher wurden. Bei Summation der biologischen Stabili­tätsergebnisse über den gesamten Zeitraum von l4 Wochen und Bildung des arithmetischen Mittels (x) der Werte (n = ll) ergibt sich das in der folgenden Tabelle dargestellte Bild, wobei ein Wert von l0,0 entsprechend l0⁸ I.E. IFN-alpha-2 pro l00 g, als l00% Aktivität gesetzt wurde:

Figure imgb0008
The stability behavior was followed over a period of 14 weeks (= 3 1/2 months). It was surprisingly and completely unexpectedly found that the stability differences became clearer with increasing storage temperature. When the biological stability results are summed over the entire period of 14 weeks and the arithmetic mean (x) of the values (n = ll) is obtained, the following table shows the picture, where a value of l0.0 corresponding to l0⁸ IE IFN-alpha-2 per 100 g was set as 100% activity:
Figure imgb0008

Aus dieser Tabelle kann man deutlich die stabilisierende Wirkung der Kombination von Hydroxyäthylcellulose und Gela­tine ablesen. Bei einer Lagerung im Kühlschrank (= plus 4°C) konnte praktisch keine Abnahme der Aktivität nach 3 l/2 Mo­naten der Formulierung mit Gelatinezusatz festgestellt wer­den, während die Formulierung ohne Gelatinezusatz einen Ab­fall von ca. 25 % zeigte. Kühlschranktemperatur stellt dabei eine praktikable Lagerung für ein Handelspräparat dar. Bei einer Lagerung bei 2l°C war ein Aktivitätsverlust der Formu­lierung ohne Gelatinezusatz innerhalb des Lagerzeitraumes von 3 l/2 Monaten auf fast die Hälfte des Ausgangswertes festzustellen, während die erfindungsgemäße Formulierung mit Hydroxyäthylcellulose und Gelatine innerhalb dieses Zeit­raumes nur einen Aktivitätsverlust von ll% aufwies, was einer relativen Stabilitätserhöhung von IFN alpha 2 von über 30% gleichkommt.This table clearly shows the stabilizing effect of the combination of hydroxyethyl cellulose and gelatin. When stored in the refrigerator (= plus 4 ° C), there was practically no decrease in activity after 3 1/2 months of the formulation with the addition of gelatin, while the formulation without the addition of gelatin showed a drop of about 25%. Refrigerator temperature represents a practicable storage for a commercial preparation. When stored at 2 l ° C, a loss of activity of the formulation without addition of gelatin was found within the storage period of 3 l / 2 months to almost half of the initial value, while the formulation according to the invention with hydroxyethyl cellulose and gelatin Within this period, there was only a loss of activity of 11%, which corresponds to a relative increase in stability of IFN alpha 2 of over 30%.

Ein weiterer Lagerungsversuch über 44 Wochen (l0 Monate) einer Zubereitungsform nach Beispiel l, abgefüllt in Alumi­niumtuben mit Innenschutzlack, zeigte im Elisatest bei La­gertemperaturen von 4°C und 2l°C keinen Aktivitätsabfall gegenüber dem Anfangswert.

Figure imgb0009
A further storage experiment over 44 weeks (10 months) of a preparation form according to Example 1, filled in aluminum tubes with an internal protective lacquer, showed no decrease in activity compared to the initial value in the Elisatest at storage temperatures of 4 ° C. and 2 ° C.
Figure imgb0009

Claims (14)

1. Pharmazeutische Zubereitungsformen für die topische Anwendung enthaltend Interferon alpha, dadurch ge­kennzeichnet, daß diese als Träger und Stabilisator für das Interferon alpha 1. Cellulosederivate, Polyvinylalkohole, Polyvinyl­pyrrolidon oder Polyacrylsäure-Polymerisate oder Mischungen davon und 2. Gelatine, sauer oder alkalisch aufgeschlossen, 1. Pharmaceutical preparation forms for topical use containing interferon alpha, characterized in that these act as carriers and stabilizers for the interferon alpha 1. Cellulose derivatives, polyvinyl alcohols, polyvinyl pyrrolidone or polyacrylic acid polymers or mixtures thereof and 2. Gelatin, acidic or alkaline digested, sowie zusätzlich ein Antiadhäsionsmittel und, gege­benenfalls, weitere übliche Hilfsstoffe enthalten.and additionally contain an anti-adhesive and, if appropriate, further customary auxiliaries. 2. Pharmazeutische Zubereitungsformen für die topische Anwendung enthaltend Interferon alpha nach Anspruch l, dadurch gekennzeichnet, daß diese ein Hydrogel sind und als Träger und Stabilisator für das Inter­feron alpha 1. Cellulosederivate, Polyvinylalkohole, Polyvinyl­pyrrolidon oder Polyacrylsäure-Polymerisate oder Mischungen davon und 2. Gelatine, sauer oder alkalisch aufgeschlossen, 2. Pharmaceutical preparation forms for topical use containing interferon alpha according to claim 1, characterized in that they are a hydrogel and as a carrier and stabilizer for the interferon alpha 1. Cellulose derivatives, polyvinyl alcohols, polyvinyl pyrrolidone or polyacrylic acid polymers or mixtures thereof and 2. Gelatin, acidic or alkaline digested, sowie zusätzlich ein Antiadhäsionsmittel und, gegebenenfalls, weitere übliche Hilfsstoffe enthalten.and additionally contain an anti-adhesive and, if appropriate, further customary auxiliaries. 3. Pharmazeutische Zubereitungsformen nach Anspruch 2, dadurch gekennzeichnet, daß diese als Träger und Stabilisator Hydroxyäthylcellulose und Gelatine ent­halten,jeweils in einer auf das fertige Produkt be­zogenen Menge von 0,l bis 2,0 Gew.%.3. Pharmaceutical preparation forms according to claim 2, characterized in that they contain as a carrier and stabilizer hydroxyethyl cellulose and gelatin, each in an amount based on the finished product from 0.1 to 2.0% by weight. 4. Pharmazeutische Zubereitungsformen nach Anspruch 2, dadurch gekennzeichnet, daß diese als Träger und Stabilisator Hydroxyäthylcellulose und Gelatine ent­halten, wobei die auf das fertige Produkt bezogene Menge an Gelatine von 0,l bis l0 Gew.% beträgt.4. Pharmaceutical preparation forms according to claim 2, characterized in that they contain as a carrier and stabilizer hydroxyethyl cellulose and gelatin, the amount of gelatin based on the finished product being from 0.1 to 10% by weight. 5. Pharmazeutische Zubereitungsformen nach einem der Ansprüche l bis 4, dadurch gekennzeichnet, daß das Antiadhäsionsmittel aus anionaktiven, kationaktiven oder neutralen oberflächenaktiven Substanzen, vor­zugsweise aus polyäthoxylierten Sorbitan-Estern be­steht.5. Pharmaceutical preparation forms according to one of claims 1 to 4, characterized in that the anti-adhesive agent consists of anionic, cationic or neutral surface-active substances, preferably of polyethoxylated sorbitan esters. 6. Pharmazeutische Zubereitungsformen nach einem der Ansprüche l bis 5, dadurch gekennzeichnet, daß das darin enthaltene Interferon alpha aus einer Zellkul­tur erhalten wird, die mit einer für Human Inter­feron alpha codierenden DNA-Sequenz transformiert wurde.6. Pharmaceutical preparation forms according to one of claims 1 to 5, characterized in that the interferon alpha contained therein is obtained from a cell culture which has been transformed with a DNA sequence coding for human interferon alpha. 7. Pharmazeutische Zubereitungsformen nach einem der Ansprüche l bis 6, dadurch gekennzeichnet, daß die­se als zusätzliche Hilfsstoffe ein Puffersystem für einen pH-Bereich von 6,0 bis 7,4, Feuchthaltesub­stanzen, hautverträgliche Konservierungsstoffe, In­terferon alpha - kompatible Penetrationsförderer und ein Vehikel enthalten.7. Pharmaceutical preparation forms according to one of claims 1 to 6, characterized in that they contain, as additional auxiliaries, a buffer system for a pH range from 6.0 to 7.4, moisturizing substances, skin-compatible preservatives, interferon alpha-compatible penetration promoters and a vehicle . 8. Pharmazeutische Zubereitungsformen nach Anspruch 7, dadurch gekennzeichnet, daß es als Feuchthaltesub­stanzen Glycerin, l,2-Propylenglykol, Sorbit, Buty­lenglykol, ein Polyol wie Polyethylenglykol 400 oder ein Gemisch davon enthält.8. Pharmaceutical preparation forms according to claim 7, characterized in that it contains glycerol, l, 2-propylene glycol, sorbitol, butylene glycol, a polyol such as polyethylene glycol 400 or a mixture thereof as moisturizing substances. 9. Pharmazeutische Zubereitungsformen nach Anspruch 7, dadurch gekennzeichnet, daß der für Interferon alpha kompatible Penetrationsförderer bis zu 50 Gew.% Di­methylsulfoxid ist.9. Pharmaceutical preparation forms according to claim 7, characterized in that the penetration promoter compatible with interferon alpha is up to 50% by weight of dimethyl sulfoxide. l0. Anwendung von pharmazeutischen Zubereitungsformen nach Ansprüchen l bis 9, dadurch gekennzeichnet, daß diese sowohl als Virusstatika im dermalen, nasa­len, ophtalmischen und intravaginalen Bereich bei Herpes labialis, Herpes genitalis inclusive Papil­loma-Infektionen, Warzen und bei Herpes Zoster-In­fektionen als auch zur topischen Behandlung von Pa­racancerosen und Cancerosen eingesetzt werden.l0. Use of pharmaceutical preparation forms according to claims 1 to 9, characterized in that they are used both as virus static in the dermal, nasal, ophthalmic and intravaginal area for herpes labialis, genital herpes including papilloma infections, warts and for herpes zoster infections as well as for topical treatment of paracanceroses and canceroses. 11. Verfahren zur Herstellung von pharmazeutischen Zu­bereitungsformen gemäß Anspruch l für die topische Anwendung mit Stabilisierung des darin enthaltenen Interferon alpha, dadurch gekennzeichnet, daß a. Gelatine, in einer auf das fertige Produkt bezo­genen Menge von 0,l bis 2,0 Gew.% in bis zu 80°C heißem Wasser gelöst wird und in diese Lösung das Antiadhäsionsmittel zugegeben wird, b. in diese Lösung gegebenenfalls Konservierungsmit­tel und Feuchthalter, Salze, Puffersysteme und Penetrationsförderer eingebracht werden, c. in diese Lösung bei bis zu 80°C Hydroxyäthylcel­lulose oder Carboxymethylcellulose, Methylcellu­lose, Hydroxypropylmethylcellulose, Polyvinylal­kohole, Polyvinylpyrrolidon, Polyacrylsäure oder ein Gemisch davon vorzugsweise in einer auf das fertige Produkt bezogenen Menge von 0,l bis 2,0 Gew.%, bei Polyvinylalkoholen oder Polyvinyl­pyrrolidon bis zu l5 Gew.%, eingebracht werden, d. Interferon alpha in einer Menge von l × l0⁵ bis l × l0¹⁰ IE/l00 g fertiges Produkt, darin homo­gen verteilt und diese Mischung steril abgefüllt wird. 11. A process for the preparation of pharmaceutical preparation forms according to claim l for topical use with stabilization of the interferon alpha contained therein, characterized in that a. Gelatin, in a quantity of 0.1 to 2.0% by weight, based on the finished product, is dissolved in hot water at up to 80 ° C. and the anti-adhesive agent is added to this solution, b. if necessary, preservatives and humectants, salts, buffer systems and penetration promoters are introduced into this solution, c. in this solution at up to 80 ° C hydroxyethyl cellulose or carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohols, polyvinyl pyrrolidone, polyacrylic acid or a mixture thereof, preferably in an amount based on the finished product of 0.1 to 2.0% by weight, in the case of polyvinyl alcohols or Polyvinylpyrrolidone up to 15% by weight, can be introduced, d. Interferon alpha in an amount of l × l0⁵ to l × l0¹⁰ IU / l00 g of finished product, homogeneously distributed therein and this mixture is filled sterile. 12. Verfahren zur Herstellung von pharmazeutischen Zu­bereitungsformen gemäß Anspruch l für die intra­vaginale Applikation, dadurch gekennzeichnet, daß a. Hydroxyäthylcellulose in einer Menge von 0,l bis 2,0 Gew.% zusammen mit Gelatine in einer Menge bis zu l0 Gew.% bei Temperaturen bis zu 60°C in einem Wasser-Glycerin-Gemisch gelöst werden, und b. bei einer Temperatur von 40 bis 50°C die Polysor­bat-haltige salzsaure IFN alpha-Lösung vorsichtig eingerührt und c. diese Lösung sofort in geeignete Formen ausgegos­sen wird. 12. A method for producing pharmaceutical preparation forms according to claim 1 for intravaginal application, characterized in that a. Hydroxyethylcellulose in an amount of 0.1 to 2.0% by weight can be dissolved together with gelatin in an amount up to 10% by weight at temperatures up to 60 ° C. in a water-glycerol mixture, and b. the polysorbate-containing hydrochloric acid IFN alpha solution is carefully stirred in at a temperature of 40 to 50 ° C. and c. this solution is immediately poured into suitable molds.
EP87100792A 1986-02-05 1987-01-21 Pharmaceutical preparations for the stabilization of interferon alpha Expired - Lifetime EP0231816B1 (en)

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EP0332222A2 (en) * 1988-03-11 1989-09-13 Teikoku Seiyaku Co., Ltd. Intravaginal delivery of biologically active polypeptides
WO1992016182A2 (en) * 1991-03-22 1992-10-01 E.B. Michaels Research Associates, Inc. Method for inactivating enveloped viruses and sperm
US5266310A (en) * 1987-09-17 1993-11-30 Boehringer Ingelheim International Gmbh Stabilization of therapeutically active proteins in pharmaceutical preparations
US5389676A (en) * 1991-03-22 1995-02-14 E. B. Michaels Research Associates, Inc. Viscous surfactant emulsion compositions
US5403579A (en) * 1986-02-25 1995-04-04 E. B. Michaels Research Associates, Inc. Process and composition for oral hygiene
WO1999064441A1 (en) * 1998-06-10 1999-12-16 Suomen Punainen Risti Veripalvelu Method for preparing virus-safe pharmaceutical compositions
WO2001066084A2 (en) * 2000-03-07 2001-09-13 Rush-Presbyterian-St. Luke's Medical Center Compositions and methods for trapping and inactivating pathogenic microbes and spermatozoa
EP1203579A1 (en) * 2000-11-03 2002-05-08 Jan Marini Skin Research Inc. Cosmetic skin care compositions containing alpha interferon
EP1449540A1 (en) * 2001-11-02 2004-08-25 Sekisui Chemical Co., Ltd. Cytokine-inducing material and cytokine-inducing instrument
CN105530950A (en) * 2013-06-09 2016-04-27 艾弗兰纳特有限公司 Compositions comprising gc- macrophage activating factor and uses thereof

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JPH07501320A (en) * 1991-10-11 1995-02-09 ギリーズ，マーク・セドリック Treatment of ocular fibrosis with interferon alpha
US5863530A (en) * 1991-10-11 1999-01-26 Spruson & Ferguson Treating ophthalmic fibrosis using interferon-α
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US5403579A (en) * 1986-02-25 1995-04-04 E. B. Michaels Research Associates, Inc. Process and composition for oral hygiene
DE3731255A1 (en) * 1987-09-17 1989-04-06 Boehringer Ingelheim Int Stabilization of therapeutically active proteins in pharmaceutical preparations
EP0307857A1 (en) * 1987-09-17 1989-03-22 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Stabilization of therapeutically active proteins in pharmaceutical preparations
US5266310A (en) * 1987-09-17 1993-11-30 Boehringer Ingelheim International Gmbh Stabilization of therapeutically active proteins in pharmaceutical preparations
EP0332222A2 (en) * 1988-03-11 1989-09-13 Teikoku Seiyaku Co., Ltd. Intravaginal delivery of biologically active polypeptides
EP0332222A3 (en) * 1988-03-11 1991-03-06 Teikoku Seiyaku Co., Ltd. Intravaginal delivery of biologically active polypeptides
US5824646A (en) * 1988-03-11 1998-10-20 Teikoku Seiyaku Co., Ltd. Intravaginal delivery of biologically active polypeptides
WO1992016182A2 (en) * 1991-03-22 1992-10-01 E.B. Michaels Research Associates, Inc. Method for inactivating enveloped viruses and sperm
US5389676A (en) * 1991-03-22 1995-02-14 E. B. Michaels Research Associates, Inc. Viscous surfactant emulsion compositions
WO1992016182A3 (en) * 1991-03-22 1992-12-23 Michaels E B Res Ass Method for inactivating enveloped viruses and sperm
WO1999064441A1 (en) * 1998-06-10 1999-12-16 Suomen Punainen Risti Veripalvelu Method for preparing virus-safe pharmaceutical compositions
WO2001066084A2 (en) * 2000-03-07 2001-09-13 Rush-Presbyterian-St. Luke's Medical Center Compositions and methods for trapping and inactivating pathogenic microbes and spermatozoa
WO2001066084A3 (en) * 2000-03-07 2002-02-28 Rush Presbyterian St Luke Compositions and methods for trapping and inactivating pathogenic microbes and spermatozoa
EP1203579A1 (en) * 2000-11-03 2002-05-08 Jan Marini Skin Research Inc. Cosmetic skin care compositions containing alpha interferon
EP1449540A1 (en) * 2001-11-02 2004-08-25 Sekisui Chemical Co., Ltd. Cytokine-inducing material and cytokine-inducing instrument
EP1449540A4 (en) * 2001-11-02 2007-01-10 Sekisui Chemical Co Ltd Cytokine-inducing material and cytokine-inducing instrument
CN105530950A (en) * 2013-06-09 2016-04-27 艾弗兰纳特有限公司 Compositions comprising gc- macrophage activating factor and uses thereof

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